Biomedical subjects

Jung-Hwa Oh

Publications and source records attributed to Jung-Hwa Oh.

10 recordsLinked to original sources

PubMed2007

Identification of genes related to Parkinson's disease using expressed sequence tags.

In a search for novel target genes related to Parkinson's disease (PD), two full-length cDNA libraries were constructed from a human normal substantia nigra (SN) and a PD patient's SN. An analysis of the gene expression profiles between them was done using the expressed sequence tags (ESTs) frequency. Data for the differently expressed genes were verified by quantitative real-time RT-PCR, immunohistochemical analysis and a cell death assay. Among the 76 genes identified with a significant difference (P > 0.9), 21 upregulated genes and 13 downregulated genes were confirmed to be differentially expressed in human PD tissues and/or in an MPTP-treated mice model by quantitative real-time RT-PCR. Among those genes, an immunohistochemical analysis using an MPTP mice model for alpha-tubulin including TUBA3 and TUBA6 showed that the protein levels are downregulated, as well as the RNA levels. In addition, MBP, PBP and GNAS were confirmed to accelerate cell death activity, whereas SPP1 and TUBA3 to retard this process. Using an analysis of ESTs frequency, it was possible to identify a large number of genes related to human PD. These new genes, MBP, PBP, GNAS, SPP1 and TUBA3 in particular, represent potential biomarkers for PD and could serve as useful targets for elucidating the molecular mechanisms associated with PD.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed2006

Identification of intrahepatic cholangiocarcinoma related genes by comparison with normal liver tissues using expressed sequence tags.

Intrahepatic cholangiocarcinoma (ICC), a malignant tumor derived from the bile duct epithelium, is one of the leading causes of death from cancer, worldwide. However, the mechanisms related to it remain largely unknown. In this study, an analysis of the gene expression profiles for ICC was done using the frequency of the ESTs obtained from nine cDNA libraries that constructed from 4 ICC cell lines and 4 normal liver tissues. One hundred and thirty-seven genes were identified as being either up- or down-regulated in human ICC cells. Thirty genes were randomly selected to confirm their differential expression in 4 human ICC cell lines and 5 ICC tissues compared to normal liver tissues by semi-quantitative RT-PCR. Among these genes, ANXA1, ANXA2, AMBP, and SERPINC1 were further verified by immunohistochemical analyses. In conclusion, these identified genes represent potential biomarkers for ICC and represent potential targets for elucidating the molecular mechanisms that are associated with ICC.

Bile Duct Neoplasms↗

PubMed2006

Gene expression profiling between embryonic and larval stages of the silkworm, Bombyx mori.

To elucidate the molecular mechanisms associated with metamorphic phenomenon relating to Bombyx mori, an important organism in the sericulture industry, we identified genes that are expressed in the different developmental stages, specifically the embryonic (ES) and larval (LS) stages of B. mori. Of 8230 high-quality ESTs from two full-length enriched cDNA libraries, 3442 of the ES ESTs were coalesced into 1325 clusters, while 4788 were coalesced into 927 clusters. The functional classification of these ESTs based on Gene Ontology showed that the types of genes that are associated with oxidoreductase activity, enzyme inhibition, and larval development were highly observed in LS, whereas the types of genes that are involved in nucleotide binding, enzyme activity, and protein transport activity were highly observed in ES. In addition, when the gene expression profile between ES and LS was examined by counting the EST frequencies in each library, 69 genes were identified as being either up- or down-regulated in the larval stage compared to the embryonic stage (P>0.99) and this was confirmed by semi-quantitative RT-PCR. The results show that genes involved in proteolysis and peptidolysis, and lipid and carbohydrate metabolism were dramatically up-regulated in LS, while those related to protein metabolism, DNA/RNA, and coenzymes were highly down-expressed. In particular, a GO analysis of these genes revealed that genes that are involved in hydrolase activity were observed to be highly expressed in amount as well as diversity in LS, while those involved in nucleic acid binding were highly expressed in ES. These data may contribute to elucidating genetic events that distinguish the developmental stage and to our understanding of the metamorphosis of B. mori.

Animals↗

PubMed2006

Generation and analysis of large-scale expressed sequence tags (ESTs) from a full-length enriched cDNA library of porcine backfat tissue.

BACKGROUND: Genome research in farm animals will expand our basic knowledge of the genetic control of complex traits, and the results will be applied in the livestock industry to improve meat quality and productivity, as well as to reduce the incidence of disease. A combination of quantitative trait locus mapping and microarray analysis is a useful approach to reduce the overall effort needed to identify genes associated with quantitative traits of interest. RESULTS: We constructed a full-length enriched cDNA library from porcine backfat tissue. The estimated average size of the cDNA inserts was 1.7 kb, and the cDNA fullness ratio was 70%. In total, we deposited 16,110 high-quality sequences in the dbEST division of GenBank (accession numbers: DT319652-DT335761). For all the expressed sequence tags (ESTs), approximately 10.9 Mb of porcine sequence were generated with an average length of 674 bp per EST (range: 200-952 bp). Clustering and assembly of these ESTs resulted in a total of 5,008 unique sequences with 1,776 contigs (35.46%) and 3,232 singleton (65.54%) ESTs. From a total of 5,008 unique sequences, 3,154 (62.98%) were similar to other sequences, and 1,854 (37.02%) were identified as having no hit or low identity (<95%) and 60% coverage in The Institute for Genomic Research (TIGR) gene index of Sus scrofa. Gene ontology (GO) annotation of unique sequences showed that approximately 31.7, 32.3, and 30.8% were assigned molecular function, biological process, and cellular component GO terms, respectively. A total of 1,854 putative novel transcripts resulted after comparison and filtering with the TIGR SsGI; these included a large percentage of singletons (80.64%) and a small proportion of contigs (13.36%). CONCLUSION: The sequence data generated in this study will provide valuable information for studying expression profiles using EST-based microarrays and assist in the condensation of current pig TCs into clusters representing longer stretches of cDNA sequences. The isolation of genes expressed in backfat tissue is the first step toward a better understanding of backfat tissue on a genomic basis.

Adipose Tissue↗

PubMed2006

Gene expression profiling of human HBV- and/or HCV-associated hepatocellular carcinoma cells using expressed sequence tags.

Liver cancer is one of the leading causes of cancer death worldwide. To identify novel target genes that are related to liver carcinogenesis, we examined new genes that are differentially expressed in human hepatocellular carcinoma (HCC) cell lines and tissues based on the expressed sequence tag (EST) frequency. Eleven libraries were constructed from seven HCC cell lines and three normal liver tissue samples obtained from Korean patients. An analysis of gene expression profiles for HCC was performed using the frequency of ESTs obtained from these cDNA libraries. Genes were identified (n=120) as being either up- or down-regulated in human liver cancer cells. Among these, 14 genes (FTL, K-ALPHA1, LDHA, RPL4, ENO1, ANXA2, RPL9, RPL10, RPL13A, GNB2L1, AMBP, GC, A1BG, and SERPINC1), in addition to previously well-known liver cancer related genes, were confirmed to be differentially expressed in seven liver cancer cell lines and 17 HCC tissues by semi-quantitative RT-PCR. In addition, 73 genes, in which there was a significant difference (P>0.99) between HBV- and HCV-associated HCC cells, were selected. Of these, expression patterns of 14 (RPLP0, AKR1C, KRT8, GPX4, RPS15, ID1, RPS21, VIM, EEF1G, EIF4A1, HLA-C, FN1, CD44, and RPS10) were confirmed by semi-quantitative RT-PCR in four of HBV- and three of HCV-associated HCC cell lines. Among those genes, an immunohistochemical analysis for ANXA2 showed that it is expressed at high levels in HCC. Using an analysis of EST frequency, the newly identified genes, especially ANXA2, represent potential biomarkers for HCC and useful targets for elucidating the molecular mechanisms associated with HCC involving virological etiology.

Biomarkers, Tumor↗

PubMed2005

Transcriptome analysis of human gastric cancer.

To elucidate the genetic events associated with gastric cancer, 124,704 cDNA clones were collected from 37 human gastric cDNA libraries, including 20 full-length enriched cDNA libraries of gastric cancer cell lines and tissues from Korean patients. An analysis of the collected ESTs revealed that 97,930 high-quality ESTs coalesced into 13,001 clusters, of which 11,135 clusters (85.6%) were annotated to known ESTs. The analysis of the full-length cDNAs also revealed that 4862 clusters (51.7%) contained at least one putative full-length cDNA clone with an initiation codon, with the average length of the 5' UTR of 140 bp. A large number appear to have a diverse transcription start site (TSS). An examination of the TSS of some genes, such as TEGT and GAPD, using 5' RACE revealed that the predicted TSSs are actually found in human gastric cancer cells and that several TSSs differ depending on the specific gastric cell line. Furthermore, of the human gastric ESTs, 766 genes (9.5%) were present as putative alternatively spliced variants. Confirmation of the predicted spliced isoforms using RT-PCR showed that the predicted isoforms exist in gastric cancer cells and some isoforms coexist in gastric cell lines. These results provide potentially useful information for elucidating the molecular mechanisms associated with gastric oncogenesis.

5' Untranslated Regions↗

PubMed2005

Identification of gastric cancer-related genes using a cDNA microarray containing novel expressed sequence tags expressed in gastric cancer cells.

PURPOSE: Gastric cancer is one of the most frequently diagnosed malignancies in the world, especially in Korea and Japan. To understand the molecular mechanism associated with gastric carcinogenesis, we attempted to identify novel gastric cancer-related genes using a novel 2K cDNA microarray. EXPERIMENTAL DESIGN: A 2K cDNA microarray was fabricated from 1,995 novel expressed sequence tags (ESTs) showing no hits or a low homology with ESTs in public databases from our 143,452 ESTs collected from gastric cancer cell lines and tissues. An analysis of the gene expression for human gastric cancer cell lines to a normal cell line was done using this cDNA microarray. Data for the different expressed genes were verified using semiquantitative reverse transcription-PCR, Western blotting, and immunohistochemical staining in the gastric cell lines and tissues. RESULTS: Forty genes were identified as either up-regulated or down-regulated genes in human gastric cancer cells. Among these, genes such as SKB1, NT5C3, ZNF9, p30, CDC20, and FEN1, were confirmed to be up-regulated genes in nine gastric cell lines and in 25 pairs of tissue samples from patients by semiquantitative reverse transcription-PCR. On the other hand, genes such as MT2A and CXX1 were identified as down-regulated genes. In particular, the SKB1, CDC20, and FEN1 genes were overexpressed in > or =68% of tissues and the MT2A gene was down-expressed in 72% of the tissues. Western blotting and immunohistochemical analyses for CDC20 and SKB1 showed overexpression and localization changes of the corresponding protein in human gastric cancer tissues. CONCLUSIONS: Novel genes that are related to human gastric cancer were identified using cDNA microarray developed in our laboratory. In particular, CDC20 and MT2A represent a potential biomarker of human gastric cancer. These newly identified genes should provide a valuable resource for understanding the molecular mechanism associated with tumorigenesis of gastric carcinogenesis and for the discovery of potential diagnostic markers of gastric cancer.

Biomarkers, Tumor↗

PubMed2004

Construction of multi-purpose vectors, pCNS and pCNS-D2, are suitable for collection and functional study of large-scale cDNAs.

To efficiently perform collection and functional studies of large-scale cDNAs, we constructed multi-functional cDNA vectors for efficient full-length cDNA cloning, direct sequencing, easy screening, and the expression of cDNA in vitro and in vivo without subcloning the cDNA into other vectors. The constructed vectors, pCNS and pCNS-D2, contain a multi-cloning site for uni-directional full-length cDNA cloning, T7 and Sp6 RNA polymerase promoters for in vitro transcription and translation, and hCMV immediate early promoter and BGH poly(A) to allow expression in mammalian cells. Using these vectors, we constructed full-length enriched cDNA libraries containing 60-75% of the full-length cDNAs using two different oligo-capping methods. The subtracted cDNA libraries could also be constructed by removing of EF1-alpha cDNA, a highly expressed cDNA. In addition, we confirmed the translation of EF1-alpha cDNA in vitro and the expression of luciferase cDNA in mammalian cells. The expression efficiency of luciferase cDNA in different cell lines, such as HeLa, Hep3B, SNU638, and SNU668, showed that pCNS vectors can highly express target genes in different cell types. These results indicated that our multi-purpose vectors, pCNS and pCNS-D2, are useful tools for the construction of full-length cDNA libraries and high-throughput based functional study of cDNAs.

Animals↗

PubMed2004

Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags.

To understand the molecular mechanism associated with gastric carcinogenesis, we identified genes expressed in gastric cancer cell lines and tissues. Of 97,609 high-quality ESTs sequenced from 36 cDNA libraries, 92,545 were coalesced into 10,418 human Unigene clusters (Build 151). The gene expression profile was produced by counting the cluster frequencies in each library. Although the profiles of highly expressed genes varied greatly from library to library, those genes related to cell structure formation, heat shock proteins, the glycolysis pathway, and the signaling pathway were highly represented in human gastric cancer cell lines and in primary tumors. Conversely, the genes encoding immunoglobulins, ribosomal proteins, and digestive proteins were down-regulated in gastric cancer cell lines and tissues compared to normal tissues. The transcription levels of some of these genes were confirmed by RT-PCR. We found that genes related to cell adhesion, apoptosis, and cytoskeleton formation were particularly up-regulated in the gastric cancer cell lines established from malignant ascites compared to those from primary tumors. This comprehensive molecular profiling of human gastric cancer should be useful for elucidating the genetic events associated with human gastric cancer.

Base Sequence↗

PubMed2003

An improved method for constructing a full-length enriched cDNA library using small amounts of total RNA as a starting material.

We have developed an improved method for constructing a full-length cDNA library using small quantity of material by modifying the original oligo-capping method. In our devised method, total RNAs are used in sequential oligo-capping steps directly without preliminary mRNA purification. Using this method, we constructed full- length cDNA libraries from 100 mg of total RNA. These libraries contained 8x10(5) to 8x10(6) independent clones with average insert sizes of 2.0 kb. Moreover, the number of full-length cDNAs containing the translation initiation codon ATG in the constructed libraries was estimated to 60-70%. In addition, 54% of the known cDNAs had a longer 5' end than the corresponding genes in the public database. Our results show that the method can be effectively used to construct full-length enriched cDNA libraries, especially, if starting material is limited.

Base Sequence↗